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We herein report a peptide receptor-based bioelectronic nose (PRBN) that can determine the quality of seafood in real-time through measuring the amount of trimethylamine (TMA) generated from spoiled seafood. The PRBN was developed using single walled-carbon nanotube field-effect transistors (SWNT-FETs) functionalized with olfactory receptor-derived peptides (ORPs) which can recognize TMA and it allowed us to sensitively and selectively detect TMA in real-time at concentrations as low as 10fM. Utilizing these properties, we were able to not only determine the quality of three kinds of seafood (oyster, shrimp, and lobster), but were also able to distinguish spoiled seafood from other types of spoiled foods without any pretreatment processes. Especially, the use of small synthetic peptide rather than the whole protein allowed PRBNs to be simply manufactured through a single-step process and to be reused with high reproducibility due to no requirement of lipid bilayers. Furthermore, the PRBN was produced on a portable scale making it effectively useful for the food industry where the on-site measurement of seafood quality is required.

There have been many trials to visualize smell using various techniques in order to objectively express the smell because information obtained from the sense of smell in human is very subjective. So far, well-trained experts such as a perfumer, complex and large-scale equipment such as GC-MS, and an electronic nose have played major roles in objectively detecting and recognizing odors. Recently, an optoelectronic nose was developed to achieve this purpose, but some limitations regarding the sensitivity and the number of smells that can be visualized still persist. Since the elucidation of the olfactory mechanism, numerous researches have been accomplished for the development of a sensing device by mimicking human olfactory system. Engineered olfactory cells were constructed to mimic the human olfactory system, and the use of engineered olfactory cells for smell visualization has been attempted with the use of various methods such as calcium imaging, CRE reporter assay, BRET, and membrane potential assay; however, it is not easy to consistently control the condition of cells and it is impossible to detect low odorant concentration. Recently, the bioelectronic nose was developed, and much improved along with the improvement of nano-biotechnology. The bioelectronic nose consists of the following two parts: primary transducer and secondary transducer. Biological materials as a primary transducer improved the selectivity of the sensor, and nanomaterials as a secondary transducer increased the sensitivity. Especially, the bioelectronic noses using various nanomaterials combined with human olfactory receptors or nanovesicles derived from engineered olfactory cells have a potential which can detect almost all of the smells recognized by human because an engineered olfactory cell might be able to express any human olfactory receptor as well as can mimic human olfactory system. Therefore, bioelectronic nose will be a potent tool for smell visualization, but only if two technologies are completed. First, a multi-channel array-sensing system has to be applied for the integration of all of the olfactory receptors into a single chip for mimicking the performance of human nose. Second, the processing technique of the multi-channel system signals should be simultaneously established with the conversion of the signals to visual images. With the use of this latest sensing technology, the realization of a proper smell-visualization technology is expected in the near future.

In the brain and central nervous system, dopamine plays a crucial role as a neurotransmitter or a local chemical messenger for interneuronal communication. Dopamine is associated with renal, hormonal, and cardiovascular systems. Additionally, dopamine dysfunction is known to cause serious illnesses, such as Parkinson's disease and Alzheimer's disease. Therefore, dopamine detection is essential for medical diagnosis and disease prevention and requires a novel strategy with high sensitivity and selectivity and a rapid response. Herein, we present a novel human dopamine receptor (hDRD1)-conjugated multidimensional conducting polymer nanofiber (NF) membrane for the selective and sensitive detection of dopamine. The membrane, which consists of multidimensional carboxylated poly(3,4-ethylenedioxythiophene) (MCPEDOT) NFs with nanorods, is used as a transistor in a liquid-ion gated field-effect transistor (FET)-based biosensor. Interestingly, hDRD1 is first expressed in Escherichia coli before it is immobilized onto the MCPEDOT NF. The hDRD1-MCPEDOT NF-based FET exhibits a rapid real-time response (2 s) with high dopamine selectivity and sensitivity performance (approximately 100 fM). Furthermore, this FET device can be integrated into a poly(dimethylsiloxane)-based microfluidic system and also can retain its high performance in the integrated system, which results in the generation of large-scale dopamine biosensors with a novel geometry.

Construction of 3-dimensional (3-D) engineered tissue is increasingly being investigated for use in drug discovery and regenerative medicine. Here, we developed multi-layered 3-D cellular assembly by using magnetic nanoparticles (MNP) isolated from Magnetospirillum sp. AMB-1 magnetotactic bacteria. Magnetized human dermal fibroblasts (HDFBs) were prepared by treatment with the MNP, induced to form 3-D assembly under a magnetic field. Analyses including LIVE/DEAD assay, transmission electron microscopy revealed that the MNP were internalized via clathrin-mediated endocytosis without cytotoxicity. The magnetized HDFBs could build 3-D structure as a function of seeding density. When the highest seeding density (5 × 105 cells/mm2 was used, the thickness of assembly was 41.90 ± 1.69 μm, with approximately 9.3 ± 1.6 cell layers being formed. Immunofluorescence staining confirmed homogeneous distribution of ECM and junction proteins throughout the 3-D assembly. Real-time PCR analysis showed decrease in expression levels of collagen types I and IV but increase in that of connexin 43 in the 3-D assembly compared with the 2-D culture. Finally, we demonstrated that the discernible layers can be formed hierarchically by serial assembly. In conclusion, our study showed that a multi-layered structure can be easily prepared using magnetically-assisted cellular assembly with highlighting cell-cell and cell-ECM communication.

Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme-degrading neutral glycosphingolipid that is transported to lysosome. Glycosphingolipid accumulation by this disease causes multi-organ dysfunction and premature death of the patient. Currently, enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. To maximize the efficacy of treatment, enhancement of cellular delivery and enzyme stability is a challenge in ERT using α-galactosidase. In this study, protein nanoparticles using human serum albumin (HSA) and 30Kc19 protein, originating from silkworm, were used to enhance the delivery and intracellular α-galactosidase stability. 30Kc19-HSA nanoparticles loaded with the α-galactosidase were formed by desolvation method. 30Kc19-HSA nanoparticles had a uniform spherical shape and were well dispersed in cell culture media. 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of delivered α-galactosidase in human foreskin fibroblast. Additionally, they showed enhanced globotriaosylceramide degradation in Fabry patients’ fibroblasts. It is expected that 30Kc19-HSA protein nanoparticles could be used as an effective tool for efficient delivery and enhanced stability of drugs.

Stem cells possess unique self-renewal and differentiation properties that make them especially well-suited for tissue engineering and regenerative medicine. According to recent studies concerning the therapeutic use of stem cells, one of the critical barriers is the regulation of lineage specific differentiation. There have been plenty of efforts to control the differentiation of the stem cells for their induction into their intended purpose. Recently, magneto-responsive materials have been used to direct stem cell differentiation, due to the strengths of the magnet-based approaches applied to stem cell research. Magnetic nanoparticles, widely used nano-sized magnetic particles, are easily controllable by a manipulated magnetic force and also their size is adequate for sensitive regulation at the cellular level. And through combination with the magnetic nanoparticles, the stem cells could be magnetized resulting in the possession of mobility by the artificial magnetic force. Therefore, by using magnetic-responsive materials and a manipulated magnetic field, it is not only stem cell differentiation efficiency but also tissue engineering adaptability, represented as transplantation efficacy, that are significantly enhanced.

Rapid and precise discrimination of various odorants is vital to fabricating enhanced sensing devices in the fields of disease diagnostics, food safety, and environmental monitoring. Here, we demonstrate an ultrasensitive and flexible field-effect transistor (FET) olfactory system, namely, a bioelectronic nose (B-nose), based on plasma-treated bilayer graphene conjugated with an olfactory receptor. The stable p- and n-type behaviors from modified bilayer graphene (MBLG) took place after controlled oxygen and ammonia plasma treatments. It was integrated with human olfactory receptors 2AG1 (hOR2AG1: OR), leading to the formation of the liquid-ion gated FET-type platform. ORs bind to the particular odorant amyl butyrate (AB), and their interactions are specific and selective. The B-noses behave as flexible and transparent sensing devices and can recognize a target odorant with single-carbon-atom resolution. The B-noses are ultrasensitive and highly selective toward AB. The minimum detection limit (MDL) is as low as 0.04 fM (10(-15); signal-to-noise: 4.2), and the equilibrium constants of OR-oxygen plasma-treated graphene (OR-OG) and ammonia plasma-treated graphene (-NG) are ca. 3.44 × 10(14) and 1.47 × 10(14) M(-1), respectively. Additionally, the B-noses have long-term stability and excellent mechanical bending durability in flexible systems.

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.

Recognition of diverse hormones in the human body is a highly significant challenge because numerous diseases can be affected by hormonal imbalances. However, the methodologies reported to date for detecting hormones have exhibited limited performance. Therefore, development of innovative methods is still a major concern in hormone-sensing applications. In this study, we report an immobilization-based approach to facilitate formation of close-packed arrays of carboxylated polypyrrole nanoparticles (CPPyNPs) and their integration with human parathyroid hormone receptor (hPTHR), which is a B-class family of G-protein-coupled receptors (GPCRs). Our devices enabled use of an electrically controllable liquid-ion-gated field-effect transistor by using the surrounding phosphate-buffered saline solution (pH 7.4) as electrolyte solution. Field-induced signals from the peptide hormone sensors were observed and provided highly sensitive and selective recognition of target molecules at unprecedentedly low concentrations (ca. 48 fM). This hormone sensor also showed long-term stability and excellent selectivity in fetal bovine serum. Importantly, the hormone receptor attached on the surface of CPPyNPs enabled GPCR functional studies; synergistic effects corresponding to increased hPTH peptide length were monitored. These results demonstrate that close-packed CPPyNP arrays are a promising approach for high-performance biosensing devices.

We developed a nanovesicle-based bioelectronic nose (NBN) that could recognize a specific odorant and mimic the receptor-mediated signal transmission of human olfactory systems. To build an NBN, we combined a single-walled carbon nanotube-based field effect transistor with cell-derived nanovesicles containing human olfactory receptors and calcium ion signal pathways. Importantly, the NBN took advantages of cell signal pathways for sensing signal amplification, enabling ≈ 100 times better sensitivity than that of previous bioelectronic noses based on only olfactory receptor protein and carbon nanotube transistors. The NBN sensors exhibited a human-like selectivity with single-carbon-atomic resolution and a high sensitivity of 1 fM detection limit. Moreover, this sensor platform could mimic a receptor-meditated cellular signal transmission in live cells. This sensor platform can be utilized for the study of molecular recognition and biological processes occurring at cell membranes and also for various practical applications such as food screening and medical diagnostics.

Previously, we reported that the expression of Bombyx mori 30Kc6 gene in Chinese hamster ovary (CHO) cells increases recombinant protein production by both inhibiting apoptosis and enhancing specific productivity. In this study, in order to gain a thorough understanding of the roles of 30Kc6 gene in antibody production, the mechanisms modulating cell apoptosis and specific productivity were investigated. 30Kc6 gene was introduced into a CHO cell line producing a chimeric anti-human CD20 monoclonal antibody. The stable expression of 30Kc6 increased cell viability and productivity by 46.7% and 3.4-folds, respectively. It was observed that the Bax translocation from cytosol to mitochondria and the cytochrome c (cyt c) release from mitochondrial intermembrane space to cytosol were repressed, which resulted in a decrease in the activation of apoptosis executioner, caspase-3. On the other hand, 30Kc6 expression increased the specific productivity by 2.3-folds. However, at the transcription level, the relative levels of heavy and light chain mRNAs increased only by 8.3% and 8.7%, respectively, which was not accountable for the observed increment in the specific productivity. Instead, the mitochondrial membrane potential was maintained and the ATP generation was stimulated. A higher ATP level could activate the mammalian target of rapamycin (mTOR), which drives the translation initiation and elongation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (4EBP1) and S6 kinase 1 (S6K1). In the 30Kc6-expressing cells, both the 4EBP1 and S6K1 were phosphorylated at higher levels, which indicated that the increased specific productivity primarily resulted from the boost of translation process. Furthermore, it was also found that the specific uptake rates of glucose and glutamine were not affected by 30Kc6 expression, demonstrating that the enhanced ATP generation and consequently maintained mTOR activity were due to 30Kc6 expression but not the different metabolic uptake rates. In conclusion, 30Kc6 expression inhibited apoptosis by repressing the Bax translocation, which down-regulated the downstream cascade responses including cyt c release and caspase-3 activation. Also, 30Kc6 expression increased the specific productivity by enhancing the translation process.

Sensing smell is a highly complex biological process, and characterizing and mimicking the interaction between the olfactory receptor (OR) protein and its ligands is extremely challenging. Herein, we report a highly sensitive and selective human nose-like nanobioelectronic nose (nbe-nose), which responds to gaseous odorants sensitively and selectively, has a signal specificity pattern similar to that in the cellular signal transduction pathway, and maintains an antagonistic behavior similar to the human nose. The human olfaction mechanism was mimicked by using carboxylated polypyrrole nanotubes (CPNTs) functionalized with human OR protein. The nbe-nose was able to detect gaseous odorants at a concentration as low as 0.02 parts-per-trillion (ppt), which was comparable to a highly trained, human expert's nose. The nbe-nose can be used scientifically for smell mechanism studies. It can be also applied to various fields that rely on smell monitoring for industrial and public purposes.

In previous studies, we reported that the 30K protein originating from the silkworm inhibited apoptosis in mammalian cells. In this work, we demonstrated that the 30Kc19 protein, which is most abundant 30K protein in silkworm hemolymph, also enhanced enzyme stability. When the recombinant 30Kc19 protein was supplemented into distilled–deionized water containing alkaline phosphatase or horseradish peroxidase, deactivation of both enzymes induced by non-buffered DDW was significantly suppressed. The increase in enzyme stability due to the presence of 30Kc19 was similar to that observed for bovine serum albumin, which is commonly used in conventional enzyme reactions. The decrease in enzyme activity due to long-term storage in different buffer systems was also inhibited by 30Kc19. The 30Kc19 protein structure was shown to play a vital role in stabilizing the enzyme. These results imply that the recombinant 30Kc19 protein hold promise for use as an additive to increase or maintain enzyme activity.

Cell-penetrating peptides (CPPs) are attractive vectors for in vivo and in vitro cellular uptake. Their use is, however, limited by insufficient understanding of their preference for a target cell. Here, a new CPP screening method is presented that uses mRNA display. After incubating the target cell lines, such as human embryonic kidney 293 (HEK 293) and HeLa cells, with an mRNA display library for 3 h at 37°C, the CPP-mRNA nucleotide conjugates were harvested. These were amplified with PCR and subsequently sequenced. The screened CPPs for each cell line were identified after four rounds of selection. Among them, two peptides, MAMPGEPRRANVMAHKLEPASLQLR NSCA (CPPK) and MAPQRDTVGGRTTPPSWGPAKAQLRNSCA (CPPL) were selected, and the FITC-labeled peptides were evaluated for their ability to penetrate cells. The screened CPPs were superior to polyarginine (R(11) ), which is widely used as a standard peptide and shows good cell penetration efficiency. Our method can be applied to other target cells for which CPPs have not yet been elucidated.

Recent results of corpus-based linguistics demonstrate that context-appropriate sentences can be generated by a stochastic constraint satisfaction process. Exploiting the similarity of constraint satisfaction and DNA self-assembly, we explore a DNA assembly model of sentence generation. The words and phrases in a language corpus are encoded as DNA molecules to build a language model of the corpus. Given a seed word, the new sentences are constructed by a parallel DNA assembly process based on the probability distribution of the word and phrase molecules. Here, we present our DNA code word design and report on successful demonstration of their feasibility in wet DNA experiments of a small scale.

Significant effort has been made in the development of an artificial nose system for various applications. Advances in sensor technology have facilitated the development of high-performance electronic and bioelectronic noses. Numerous articles describe the advantages of artificial nose systems for biomedical applications. Recent advances in the development of electronic and bioelectronic noses and their biomedical applications are reviewed in this article.

The primary goal of this study was to develop a new strategy to inactivate bacterial biofilms using the thermal stress derived from superparamagnetic iron oxide nanoparticles (SPIONs) in an alternating current (AC) magnetic field. A large number of studies have examined the inactivation of bacterial biofilms using antimicrobial agents; however, there have been no attempts to inactivate biofilms by hyperthermia using SPIONs. In this study, a SPION solution was added to Pseudomonas aeruginosa (P. aeruginosa) PA01 biofilm, and heat was generated by placing the nanoparticle-containing biofilm in an AC magnetic field. The heating temperature was dependent on the concentration of the added SPION solution. More than 4 log inactivation of the PA01 biofilm was obtained using a 60 mg mL(-1) SPION solution in 8 min, and this resulted in a dramatic disintegration of the bacterial cell membrane in the biofilm. This inactivation was largely due to the thermal effect. Local heating of a specific area is also possible using this method, and the heating temperature can be easily adjusted by controlling the concentration of the SPION solution. Therefore, hyperthermia using magnetic nanoparticles holds promise as an effective tool for inactivating the bacterial biofilm.

Over-expression of anti-apoptotic cloned-genes is a widely used strategy for inhibiting apoptosis in mammalian cell culture. In our previous study, we reported Bombyx mori 30K gene improved the production of recombinant proteins in Chinese hamster ovary (CHO) cells. In this study, we reengineered the CHO cells with the 30Kc6 gene and 30Kc19 gene for the production of a therapeutic monoclonal antibody (mAb) directed against the glycoprotein receptor of human platelets. After the medium was changed from serum containing one to serum-free one, expression of 30Kc6 in CHO cells increased the cell viability by 40.8% in 4 days and mAb production by 2.3-fold in 5 days. However, no significant changes in cell viability and mAb production were observed for the cells expressing 30Kc19. In the case of the cells expressing 30Kc6, the specific production rate was also improved. The expression of the 30Kc6 gene increased the cell viability and productivity because it maintained the mitochondrial membrane potential (MMP) and reduced the downstream cascade responses for apoptosis. These results indicate that 30Kc6 outperformed 30Kc19 in terms of cell death-protective capability and the production of monoclonal antibodies in CHO cells.

The market for protein-drugs has steadily increased due their increased use as alternatives to traditional small molecule drugs. While some therapeutic proteins have been produced in microbial systems, mammalian cell systems such as Chinese hamster ovary (CHO) cells are widely used as the host cell system. To increase the efficiency of producing therapeutic proteins, many researchers have attempted to solve the critical problems that occur in mammalian cell systems. As a result, several serum-free media and advanced culture methods have been developed, and protein productivity has increased considerably through the development of efficient selection methods. However, the prevalence of apoptosis during mammalian cell culture still remains a significant problem. Based on the understanding of apoptotic mechanisms and related proteins, anti-apoptotic engineering has steadily progressed. In this study, we review the strategies that have been developed for high-level production of recombinant proteins in the CHO cell system via a selection of clones, target-gene amplification, optimization of culture systems and an inhibition of apoptosis through genetic modification.

In this study, we used a bacteriophage λQ − S − mutant that increased the stability of recombinant Escherichia coli during continuous culture. The operation was conducted in two stages: the first stage was carried out to promote cell growth, and the second stage was performed for product formation. The productivity of recombinant proteins depends on the substrate concentration of the fresh medium supplied to the second stage (S 3) and dilution rate of the second stage (D 2). With the optimal value of S 3 and D 2, the first and second stages were stably maintained for 170 and 80 h, respectively. To further improve this process, a three-stage continuous process was conducted with an additional induction stage between the growth and production stages. Compared with the two-stage operation, the stable production period was extended by 1.7 fold, and the recombinant protein production increased by 1.3 fold.

Recent progress in molecular computation suggests the possibility of pattern classification in vitro. Weighted sum is a primitive operation required by many pattern classification problems. Here we present a DNA-based molecular computation method for implementing the weighted-sum operation and its use for molecular pattern classification in a test tube. The weights of the classifier are encoded as the mixing ratios of the differentially labeled probe DNA molecules, which are competitively hybridized with the input-encoding target molecules to compute the decision boundary of classification. The computation result is detected by fluorescence signals. We experimentally verify the underlying weight encoding scheme and demonstrate successful discrimination of two-group labels of synthetic DNA mixture patterns. The method can be used for direct computation on biomolecular data in a liquid state.

The olfactory system has the ability to discriminate and identify thousands of odorant compounds at very low concentrations. Recently, many researchers have been trying to develop artificial sensing devices that are based on the olfactory system. A bioelectronic nose, which uses olfactory receptors (ORs) as sensing elements, would benefit naturally optimized molecular recognition. Accordingly, ORs can be effectively used as a biological element in bioelectronic noses. Bioelectronic nose can be classified into cell-based and protein-based biosensors. The cell-based biosensor uses living cells that express olfactory receptors as the biological sensing elements and the protein-based biosensor uses the olfactory receptor protein. The binding of odorant molecules to the ORs can be measured using various methods such as piezoelectric, optic, and electric devices. Thus, bioelectronic nose can be developed by combining the biological sensing elements with these non-biological devices. The application of bioelectronic nose in a wide range of different scientific and medical fields is essentially dependent on the development of highly sensitive and selective biosensors. These sensor systems for the rapid detection of specific odorants are crucial for environmental monitoring, anti-bioterrorism, disease diagnostics, and food safety. In this article, we reviewed recent advances in the development of bioelectronic nose.

Enterobacter asburiae SNU-1 harvested after cultivation was used as a whole cell biocatalyst, for the production of hydrogen. Formic acid was efficiently converted to hydrogen using the harvested cells with an initial hydrogen production rate and total hydrogen production of 491 ml/l/h and 6668 ml/l, respectively, when 1 g/l of whole cell enzyme was used. Moreover, new pH-stat fed-batch operation was conducted, and total hydrogen production was 1.4 times higher than that of batch operation. For practical application, bio-hydrogen produced from formic acid using harvested cells was directly applied to PEMFC for power generation.

The silkworm hemolymph has an anti-apoptotic activity in insect, mammalian, and human cell systems. The protein from silkworm hemolymph with the highest apoptosis inhibiting activity was found to be 30Kc19 protein, which was one of the ‘30K proteins’. In this study, 30Kc19 protein encoded by the 30Kc19 gene of the silkworm was expressed in Escherichia coli with (pET-22b(+)) and without (pET-3a) pelB leader sequence. 30Kc19 protein was over-expressed largely as a soluble form by pET-3a and both as soluble and insoluble forms by pET-22b(+). The medium was supplemented with each of the recombinant 30Kc19 proteins, and their presence was found to inhibit nuclear fragmentation and apoptotic body formation in actinomycin D-induced Sf9 cell apoptosis. Moreover, 30Kc19 protein repressed the activation of Sf-caspase-1. The 30Kc19 protein obtained from periplasm showed the most effective anti-apoptotic activity. This protein holds great potential for industrial and pharmaceutical applications since mass production and easy purification of this protein is possible.

We designed a wavelength interrogation-based surface plasmon resonance (SPR) biosensor to detect avian influenza DNA (AI-DNA). Hybridization reactions between target AI-DNA probes and capture probes immobilized on a gold surface were monitored quantitatively by measuring the resonance wavelength in the visible waveband. The experimental results were consistent with numerical calculations. Although the SPR detection technique does not require the DNA to be labeled, we also evaluated fluorescently-labeled targets to verify the hybridization behavior of the AI-DNA. Changes in resonance were found to be linearly proportional to the amount of bound analyte. A wavelength interrogation-type SPR biosensor can be used for rapid measurement and high-throughput detection of highly pathogenic AI viruses.

Odorant-binding proteins (OBPs) primarily function in the transport of hydrophobic odorants. In this study, OBPs originating from rat and pig were cloned into a mammalian expression vector, pcDNA3, and expressed in HEK-293 cells, and their specificity for odorants and olfactory receptors was examined. Results suggest that OBPs have a high affinity for the olfactory receptors when both the OBP and receptor originate from the same species. The rat OBPs were bound not only to the rat olfactory receptor I7 but also to the odorant specific to I7. The solubility of the odorant was increased by both OBP2 and OBP3, which originate from rat, but with different efficiencies. These results demonstrate that OBPs specifically interact with odorants as well as olfactory receptors, and these interactions can influence the sensitivity of olfactory receptor-based biosensors.

Olfactory receptors pertaining to G protein-coupled receptor (GPCR) are integral membrane proteins composed of seven transmembrane spanning domains. It has been reported that these receptor proteins are difficult to overexpress, solubilize, and purify because of their complicated structures and strong hydrophobicity. In this study, full-length human olfactory receptor (hOR) 2AG1 was overexpressed in E. coli as a fusion protein with a glutathione S-transferase (GST) tag mainly as an inclusion body without any mutations or deletions in the gene. This protein was difficult to solubilize with detergents and chaotropic agents, and only N-lauroyl sarcosine was found to be suitable for solubilizing it. In contrast, Trition X-100 was found to solubilize most of the impurity proteins from the insoluble fraction in E. coli. Based on this observation, we applied a simple and efficient column-free method using these two detergents for the purification of the olfactory receptor protein. In this method, the insoluble fraction of the cell lysate was first treated with Triton X-100 to remove impurity proteins. The remaining insoluble fraction was then further treated with N-lauroyl sarcosine to solubilize the olfactory receptor protein. Milligram quantity of the human olfactory receptor was produced. This is the first report to produce full-length of the olfactory receptor from E. coli.

Although chemically defined media have been developed and widely used to study the expression of virulence factors in the model plant pathogen Pseudomonas syringae , it has been difficult to link specific medium components to the induction response. Using a chemostat system, we found that iron is the limiting nutrient for growth in the standard hrp -inducing minimal medium and plays an important role in inducing several virulence-related genes in Pseudomonas syringae pv. tomato DC3000. With various concentrations of iron oxalate, growth was found to follow Monod-type kinetics for low to moderate iron concentrations. Observable toxicity due to iron began at 400 μM Fe 3+ . The kinetics of virulence factor gene induction can be expressed mathematically in terms of supplemented-iron concentration. We conclude that studies of induction of virulence-related genes in P. syringae should control iron levels carefully to reduce variations in the availability of this essential nutrient.

Apolipoprotein A-1 (apoA-I) forms a discoidal membrane structure with phospholipids during the formation of high density lipoprotein and plays a role in the reverse cholesterol transport pathway. The discoidal membrane nanostructure, Nanodisc, can be easily assembled in vitro using apoA-I protein and phospholipids. In this study, the possibility of exploiting this unique membrane structure for the immobilization of membrane proteins on solid surfaces while the proteins are embedded in the membrane was investigated. By using His6-tagged full-length apoA-I, a surface-attachable, membrane-protein-anchored membrane (SAMPAM) structure, in which membrane proteins of interest are embedded into the membrane, was reconstituted. When the SAMPAM was immobilized on a Ni-NTA surface, the structure maintained its size and shape, indicating that the new proposed architecture may be useful for the display of membrane proteins on a solid surface in a membrane-associated form.

Get a whiff of this: Human olfactory receptor (hOR)-conjugated polypyrrole (PPy) nanotubes were integrated into the field-effect transistor (FET) sensor platform for the fabrication of high-performance bioelectronic noses (see picture, S = source, D = drain). The device can translate and amplify hOR-odorant interaction into a detectable signal, and it showed highly sensitive and specific responses toward a target odorant.

The Bombyx mori 30Kc gene is known to have anti-apoptotic activity and can enhance the cell growth and expression of recombinant proteins in anchorage-dependent CHO cell cultures. In this study, an interferon-β (IFN-β)-producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed to investigate the effect of 30Kc6 expression on the production of IFN-β in serum-free suspension culture. The 30Kc6 expressing cell line showed lower apoptotic activity and prolonged cell viability under apoptotic conditions induced by the addition of sodium butyrate, staurosporine, or the removal of serum. The 30Kc6 expressing cell line also suppressed the loss of mitochondrial membrane potential induced under these conditions. It was observed that viability, and production of IFN-β were also enhanced by 30Kc6 expression in serum-free suspension cultures. These results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.

This work was supported by the System 2010 program of the Ministry of Knowledge Economy. SH acknowledges the support from NRL (ROA-2004-000-10438-0) and NSI-NCRC program of KOSEF. Supporting Information is available online at Wiley InterScience or from the author.

A common problem with the in vivo therapeutic applications of cells is that cells can rapidly disappear into the circulatory system after an injection. Magnetic nanoparticles can be used to solve this problem. Bacterial magnetic nanoparticles were used in this study for targeting stem cells at a specific location within a microfluidic channel. Magnetic nanoparticles were isolated from Magnetospirillum sp. AMB-1 and delivered to endothelial progenitor cells (EPCs). Cellular uptake of magnetic nanoparticles and their functional feasibility was characterized in vitro. The environment of a human blood vessel was simulated using a microfluidic channel. Magnetic nanoparticle-incorporated EPCs were injected into a microchannel and the flow rate of cells was uniformly controlled by use of a syringe pump. EPCs were effectively targeted to a specific location within the microchannel by an external magnetic field (about 400 mT). About 40% of EPCs were efficiently targeted with a flow rate of 5 microl min(-1) when 10 microg of magnetic nanoparticles were used per 10(4) cells. This microfluidic system provides a useful tool towards a better understanding of the behavior of magnetic nanoparticle-incorporated cells within the human circulatory system for clinical use.

In a two-phase operation, E. coli containing λSNNU1 (Q − S −) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.

Odorant-binding proteins are low molecular weight, soluble proteins that are secreted by glands of the nasal cavity. Their function is known to be the transport of hydrophobic odorants. This feature is important to artificial olfactory biosensors, which operate in the aqueous phase. In this study, one of rat odorant-binding proteins, OBP3, was inserted into a mammalian expression vector pcDNA3, expressed, and secreted from human embryonic kidney-293 (HEK-293) cells. The his6 tag and signal peptide of the prelysozyme (plys) were fused with OBP3 for the detection and secretion of the proteins, respectively. The secretion level of OBP3 was maximal at 3 h of incubation time. The secreted OBP3 increased the solubility of a hydrophobic odorant, octanal, which is the specific odorant of rat olfactory receptor I7. The secreted OBP3 also bound to olfactory receptor I7. These interactions consequently increased the cellular signal intensity stimulated by the odorant in the cells expressing olfactory receptor I7. Our findings indicate that odorant-binding protein can be effectively used to increase the sensitivity of olfactory receptor-based biosensors.

Apoptosis is a barrier to maintaining high viable cell densities in animal cell culture. Silkworm hemolymph and its 30K protein have been reported to exhibit anti-apoptotic activity in various mammalian and insect cell systems. The 30K protein is thermally unstable at temperatures higher than 60 degrees C; however, the silkworm hemolymph heat-treated at 70-80 degrees C still exhibited anti-apoptotic activity. This indicates that silkworm hemolymph contains another anti-apoptotic compound other than 30K protein. In this article, the anti-apoptotic molecule other than 30K protein was found from the silkworm hemolymph and identified. This molecule was storage-protein 2 (SP2), which has no homology with any known anti-apoptotic protein. This molecule was heat-stable up to 80 degrees C, while 30K protein lost its activity at temperatures higher than 60 degrees C. When apoptosis was induced by staurosporine in HeLa cells, SP2 protein suppressed nuclear fragmentation and apoptotic body formation. Moreover, the generation of reactive oxygen species after apoptosis induction was inhibited, which means the inhibition occurred in an early step of the apoptotic process. Inhibition of apoptosis by the SP2 protein would lead to the minimization of cell death during commercial mammalian cell culture.

This paper reports a physical and chemical surface modification technique to achieve a high tethering efficiency as well as controllability and coordinating bacterial cells. This technique was used to experimentally show multiple spin actuators, using the flagellar motion of AMB-1 bacteria. For physical surface modification, a polydimethylsiloxane (PDMS) pillar array, using a soft-lithography technique, was used. For chemical surface modification, a UV-crosslinked azido benzoic acid (ABA) modified surface was used. A high rate of tethering and adhesion of AMB-1 bacterial cells was achieved on the modified surface, and multiple spin actuation and motoring were observed.

Three λ mutants were constructed based on the Q − mutant in order to enhance their productivity and stability in an Escherichia coli /bacteriophage λ system. The newly constructed bacteriophage mutants named λSNU1, λSNU2, and λSNU3 were Q − S − , Q − W − E − , and Q − S − W − E − mutants, respectively. Compared to all of the mutants, λSNU1 turned out to be the best with regards to higher protein expression and better genetic stability. Mechanisms by which these attributes are achieved have been discussed. The high productivity of P90c/λSNU1 for the recombinant protein was due to the high copy number of λ DNA and high translational efficiency. This mutant phage λSNU1 can be used to provide a high level of stability and productivity of the cloned gene particularly for long-term continuous operation.

A new fermentative hydrogen-producing bacterium was isolated from a domestic landfill and identified as Enterobacter asburiae using 16S rRNA gene sequencing and DNA–DNA hybridization methods. The isolated bacterium, designated as Enterobacter asburiae SNU-1, is a new species that has never been examined as a potential hydrogen-producing bacterium. This study examined the hydrogen-producing ability of Enterobacter asburiae SNU-1. During fermentation, the hydrogen was mainly produced in the stationary phase. The hydrogen yield based on the formate consumption was 0.43 mol hydrogen/mol formate. This strain was able to produce hydrogen over a wide range of pH (4–7.5), with the optimum pH being pH 7. The level of hydrogen production was also affected by the initial glucose concentration, and the optimum value was found to be 25 g glucose/l. The maximum and overall hydrogen productivities were 398 and 174 ml/l/hr, respectively, at pH 7 with an initial glucose concentration of 25 g/l. This strain could produce hydrogen from glucose and many other carbon sources such as fructose, sucrose, and sorbitol.

Many attempts have been made to develop a serum-free medium on account of the problems caused by serum in mammalian cell culture. However, serum deprivation inhibits cell growth and induces apoptosis. Moreover, adapting host cells to the serum-free medium is difficult and time-consuming. In a previous study, the anti-apoptotic 30K proteins were identified from silkworm hemolymph, which suggests that the 30K genes coding for the anti-apoptotic compound can be used for the anti-apoptosis engineering of mammalian cells. In this study, the 30K genes ( 30Kc6 , 30Kc19 , and 30Kc123 ) were introduced to DG44 CHO cells, which are the mammalian cell line most commonly used by industry for the production of biopharmaceuticals, in order to make them resistant to the apoptosis induced by serum deprivation. Among the 30K genes, the 30Kc6 gene exhibited the highest apoptosis-inhibition activity. When the 30Kc6 -expressing cells cultivated in the serum-containing medium were transferred directly to commercially available serum-free media, 30Kc6 expression increased the viable cell density by four-fold through inhibiting serum deprivation-induced apoptosis.

One important aspect of nanotechnology includes thin films capable of being applied to a wide variety of surfaces. Indispensable functions of films include controlled surface energy, stability, and biocompatibility in physiological systems. In this study, we explored the ancient Asian coating material "lacquer" to enhance the physiological and mechanical stability of nanofilms. Lacquer is extracted from the lacquer tree and its main component called urushiol, which is a small molecule that can produce an extremely strong coating. Taking full advantage of layer-by-layer assembly techniques, we successfully fabricated urushiol-based thin films composed of small molecule/polymer multilayers by controlling their molecular interaction. Unique cairnlike nanostructures in this film, produced by urushiol particles, have advantages of intrinsic hydrophobicity and durability against mechanical stimuli at physiological environment. We demonstrated the stability tests as well as the antimicrobial effects of this film.

Transcription factors have been studied as an important drug candidate. Ever since the successful generation of induced pluripotent stem cells (iPSCs), there has been tremendous interest in reprogramming transcription factors. Because of the safety risks involved in a virus-based approach, many researchers have been trying to deliver transcription factors using nonintegrating materials. Thus, delivery of transcription factors produced as recombinant proteins in E. coli was proposed as an alternative method. However, the low level of soluble expression and instability of such recombinant proteins are potential barriers. We engineered a Bombyx mori 30Kc19 protein as a fusion partner for transcription factors to overcome those problems. We have previously reported that 30Kc19 protein can be produced as a soluble form in E. coli and has a cell-penetrating property and a protein-stabilizing effect. Transcription factors fused with 30Kc19 (Oct4-30Kc19, Sox2-30Kc19, c-Myc-30Kc19, L-Myc-30Kc19, and Klf4-30Kc19) were produced as recombinant proteins. Interestingly, Oct4 and L-Myc were expressed as a soluble form by conjugating with 30Kc19 protein, whereas Oct4 alone and L-Myc alone aggregated. The 30Kc19 protein also enhanced the stability of transcription factors both in vitro and in cells. In addition, 30Kc19-conjugated transcription factors showed rapid delivery into cells and transcriptional activity significantly increased. Overall, 30Kc19 protein conjugation simultaneously enhanced soluble expression, stability, and transcriptional activity of transcription factors. We propose that the conjugation with 30Kc19 protein is a novel approach to solve the technical bottleneck of gene regulation using transcription factors.

Monodispersed magnetite (Fe3O4) nanoparticles (NPs) were prepared through the thermal decomposition method. The obtained NPs were surface modified with silica (SiO2) and polyethylene glycol (PEG), to enhance their stability in aqueous environment and their cellular uptake efficiency for biomedical applications. The NPs were characterized by X-ray diffraction (XRD), high-resolution transmission electron microscopy (HR-TEM), Fourier transform infrared (FT-IR) spectroscopy, and dynamic light scattering (DLS). The cytotoxicity of these NPs on bone marrow mesenchymal stem cells (BM-MSCs) was measured by MTT assay (cell viability test) at various concentrations (2, 5, 12.5, 25, and 50 µg/mL). The cells remained more than 90% viable at concentrations as high as 50 µg/mL. To compare the cellular uptake efficiency, these NPs were treated in BM-MSCs and the Fe concentration within the cells was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis. The uptake process displayed a time- and dose-dependency. The uptake amount of SiO2-coated Fe3O4 (Fe3O4@SiO2) NPs was about 10 times higher than that of the PEG-coated ones (Fe3O4@PEG).

Human sensory-mimicking systems, such as electronic brains, tongues, skin, and ears, have been promoted for use in improving social welfare. However, no significant achievements have been made in mimicking the human nose due to the complexity of olfactory sensory neurons. Combinational coding of human olfactory receptors (hORs) is essential for odorant discrimination in mixtures, and the development of hOR-combined multiplexed systems has progressed slowly. Here, we report the first demonstration of an artificial multiplexed superbioelectronic nose (MSB-nose) that mimics the human olfactory sensory system, leading to high-performance odorant discriminatory ability in mixtures. Specifically, portable MSB-noses were constructed using highly uniform graphene micropatterns (GMs) that were conjugated with two different hORs, which were employed as transducers in a liquid-ion gated field-effect transistor (FET). Field-induced signals from the MSB-nose were monitored and provided high sensitivity and selectivity toward target odorants (minimum detectable level: 0.1 fM). More importantly, the potential of the MSB-nose as a tool to encode hOR combinations was demonstrated using principal component analysis.

Various attempts have been made to mimic the human olfactory system using human olfactory receptors (hORs). In particular, OR-expressed cell-based odorant detection systems mimic the smell sensing mechanism of humans, as they exploit endogenous cellular signaling pathways. However, the majority of such cell-based studies have been performed in the liquid phase to maintain cell viability, and liquid odorants were used as detection targets. Here, we present a microfluidic device for the detection of gaseous odorants which more closely mimics the human olfactory system. Cells expressing hOR were cultured on a porous membrane. The membrane was then flipped over and placed between two compartments. The upper compartment is the gaseous part where gaseous odorants are supplied, while the lower compartment is the aqueous part where viable cells are maintained in the liquid medium. Using this simple microfluidic device, we were able to detect gaseous odorant molecules by a fluorescence signal. The fluorescence signal was generated by calcium influx resulting from the interaction between odorant molecules and the hOR. The system allowed detection of gaseous odorant molecules in real-time, and the findings showed that the fluorescence responses increased dose-dependently in the range of 0-2 ppm odorant. In addition, the system can discriminate among gaseous odorant molecules. This microfluidic system closely mimics the human olfactory system in the sense that the submerged cells detect gaseous odorants.

Over the past several years, the preparation of functionalized nanoparticles has been aggressively pursued in order to develop desired structures, compositions, and structural order. Among the various nanoparticles, iron oxide magnetic nanoparticles (MNPs) have shown great promise because the material generated using these MNPs can be used in a variety of biomedical applications and possible bioactive functionalities. In this study, we report the development of various functionalized MNPs (F-MNPs) generated using the layer-by-layer (LbL) self-assembly method. To provide broad functional opportunities, we fabricated F-MNP bio-toolbox by using three different materials: synthetic polymers, natural polymers, and carbon materials. Each of these F-MNPs displays distinct properties, such as enhanced thickness or unique morphologies. In an effort to explore their biomedical applications, we generated basic fibroblast growth factor (bFGF)-loaded F-MNPs. The bFGF-loaded F-MNPs exhibited different release mechanisms and loading amounts, depending on the film material and composition order. Moreover, bFGF-loaded F-MNPs displayed higher biocompatibility and possessed superior proliferation properties than the bare MNPs and pure bFGF, respectively. We conclude that by simply optimizing the building materials and the nanoparticle's film composition, MNPs exhibiting various bioactive properties can be generated.

Olfactory receptor (OR) genes are considered to be the largest superfamily of the mammalian genome, and in the case of humans, approximately 390 kinds of functional ORs play a role in perceiving odors. In spite of their significance in olfaction, the function of all ORs has not yet been fully revealed. In order to efficiently identify specific ligands of orphan ORs, methods that can generate olfactory signals in a reliable manner and that can convert the cellular signals into measurable responses are required. Here, we describe an OR screening assay method using olfactory sensors that are based on cell-derived nanovesicles combined with single-walled carbon nanotube field-effect transistors (SWNT-FETs). The nanovesicles contain ORs on their surface membrane and induce influx of calcium ions similar to olfactory signal transduction. This ion influx causes an electrical current change along the carbon nanotube, and then this change is measured by the SWNT-FET sensor. This technique facilitates the simple and rapid screening of OR functions.